Single-base mapping of m⁶A by an antibody-independent method

N ⁶-methyladenosine (m⁶A) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput m⁶A identification method depends on the anti-m⁶A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of m⁶A. Here, we developed a precise and high-throughput antibody-independent m⁶A identification method based on the m⁶A-sensitive RNA endoribonuclease recognizing ACA motif (m⁶A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m⁶A-REF-seq). Whole-transcriptomic, single-base m⁶A maps generated by m⁶A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual m⁶A sites, confirming the high reliability and accuracy of m⁶A-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that m⁶A sites are conserved with single-nucleotide specificity and tend to cluster among species.